Antibody response after first and second BNT162b2 vaccination to predict the need for subsequent injections in nursing home residents

We explored antibody response after first and second BNT162b2 vaccinations, to predict the need for subsequent injections in nursing home (NH) residents. 369 NH residents were tested for IgG against SARS-CoV-2 Receptor-Binding Domain (RBD-IgG) and nucleoprotein-IgG (SARS-CoV-2 IgG II Quant and SARS-CoV-2 IgG Alinity assays, Abbott Diagnostics). In NH residents with prior SARS-CoV-2 infection, the first dose elicited high RBD-IgG levels (≥ 4160 AU/mL) in 99/129 cases (76.9%), with no additional antibody gain after the second dose in 74 cases (74.7%). However, a low RBD-IgG level (< 1050 AU/mL) was observed in 28 (21.7%) residents. The persistence of nucleoprotein-IgG and a longer interval between infection and the first dose were associated with a higher RBD-IgG response (p < 0.0001 and p = 0.0013, respectively). RBD-IgG below 50 AU/mL after the first dose predicted failure to reach the antibody concentration associated with a neutralizing effect after the second dose (≥ 1050 AU/mL). The BNT162b2 vaccine elicited a strong humoral response after the first dose in a majority of NH residents with prior SARS-CoV-2 infection. However, about one quarter of these residents require a second injection. Consideration should be given to immunological monitoring in NH residents to optimize the vaccine response in this vulnerable population.


Scientific Reports
| (2022) 12:13749 | https://doi.org/10.1038/s41598-022-18041-x www.nature.com/scientificreports/ SARS-CoV-2 naïve residents. The first dose of mRNA vaccine induces a strong RBD-IgG response in individuals with prior COVID-19 9,10 . Given that older adults may have a limited vaccine response, NH residents in France received two vaccine doses regardless of any prior SARS-CoV-2 infection. However, most NH residents with evidence of previous infection showed a strong humoral response after a single BNT162b2 dose, suggesting that recommendations for immunocompetent individuals could also be applied to this population 11 . Conversely, a lack of or low antibody vaccine response was observed in NH residents without prior SARS-CoV-2 infection 11,12 . Authorization for the use of the two mRNA vaccines encoding SARS-CoV-2-spike was based on the results of phase 3 clinical trials 13,14 . While these vaccines have proven high levels of efficacy in preventing severe forms of illness in the general population, it has yet to be evaluated whether immune responses elicited by SARS-CoV-2 mRNA vaccines are homogenously robust in the elderly 15,16 .
It remains unclear if the humoral response after one BNT162b2 dose is predictive of the response subsequent to two doses in older adults, and how natural immunization and the interval since infection may modulate vaccine response. A better understanding of the consequences of SARS-CoV-2 infection history and of antibody response to the first vaccine dose is needed in order to adjust vaccine policies for this vulnerable population.
In this study, in a large cohort of NH residents, we assessed the value of RBD-IgG levels after the first BNT162b2 dose to predict: (i) the lack of additional benefits from a second dose as to the generation of antibodies, (ii) a significant antibody response after the second dose, and (iii) a prior SARS-CoV-2 infection. We also assessed the impact on vaccine response of natural serological status against SARS-CoV-2 nucleocapsid, and of the interval since infection.

Methods
Design of the study. All experiments were performed in accordance with relevant guidelines and regulations. Our study is part of the CONsort-19 cohort 10,17-19 a longitudinal follow-up of NH residents performed during the COVID-19 pandemic. In this study, we monitored IgG against Receptor-Binding Domain (RBD-IgG) 3 weeks after the first BNT162b2 dose, and 6 weeks after the second dose, considered as a period in which the antibody decay rate response is stabilized 20 . Results in residents without prior COVID-19 vs. residents with prior COVID-19 were compared. Two periods were considered for COVID-19 recovery-the past 9-12 months (older infections), and the past 3-7 months (newer infections)-corresponding to the first and second waves of the epidemic in our region. RBD-IgG levels before vaccination and their relationship to vaccine response were analyzed in a subgroup of residents. We also assessed IgG levels against N-protein 6 weeks after a second BNT162b2 dose in residents infected in the previous 9-12 months vs. the previous 3-7 months, and we evaluated the impact of N-IgG status on RBD-IgG vaccine response.
Setting and participants. Invited to participate in the study were residents from nine NHs which had suffered COVID-19 outbreaks in 2020, and who were already included in the CONsort-19 cohort [17][18][19] . Residents were included after having signed an informed consent. Participants received the first dose of the BNT162b2 vaccine between January and March 2021, and received the second dose 3 weeks later. Blood collections were performed 3 weeks after the first dose and 6 weeks after the second dose. In accordance with the regional Health Agency (Occitanie, South of France) and the European Geriatric Medicine Society guidance 17 , as soon as a case of COVID-19 was diagnosed in a NH, all residents and healthcare professionals were repeatedly tested using PCR on nasopharyngeal swabs until no new cases were diagnosed. Residents were grouped by date of PCR positivity (wave 1 and 2). Residents tested negative by PCR but positive for SARS-CoV-2 nucleocapsid IgG the day before the second dose were also considered as having a prior infection without ruling on the occurrence of the SARS-CoV-2 infection during the first or second wave. The study was approved by the Montpellier University Hospital institutional review board (IRB-MTP_2020_06_202000534 and IRB-MTP _2021_04_202000534).
Laboratory methods. S-protein IgG against the SARS-CoV-2 Receptor-Binding Domain (RBD) of the S1 subunit was quantified using the SARS-CoV-2 IgG II Quant assay, a chemiluminescent microparticle test using the Alinity i automatic analyzer (Abbott Diagnostics). The Abbott IgG II method provides results linearity over a wide concentration range with good agreement with external calibration materials 21 . Results were expressed as arbitrary units per mL (AU/mL; positive threshold: 50 AU/mL; upper limit: 40,000 AU/mL). RBD-IgG levels in AU/mL can be converted in Binding Antibody Units (BAU) following manufacturers' recommendations (AU/ mL × 0.142). Serum dilutions were performed to quantify RBD-IgG when the results surpassed the higher limit of quantification of the assay. According to previous studies and manufacturer's instructions, a first threshold ≥ 1050 AU/mL was considered as a significant response 22 , and a second threshold ≥ 4160 AU/mL as a level indicating a high neutralizing effect 23 . We performed microneutralization assays to confirm these thresholds. A panel of 15 sera was tested against SARS-CoV-2 and a B.1.1.7 variant (Alpha VOC). RBD-IgG concentration was significantly correlated with neutralizing antibody titers (Spearman correlation coefficients of R 2 = 0.89 for wild type strain and 0.90 for B.1.1.7 (p < 0.0001) (Supplemental Fig. 1). As regards RBD-IgG levels, neutralizing activities were (i) high when over 4160 AU/mL (neutralizing antibody titer of 640 log IC50 (IQR: 640-1960) for wild type SARS-CoV-2, 640 log IC50 (IQR: 640-1920) for B.1.1.7 strain), (ii) absent or moderate when between 1050 and 4160 AU/mL, and (iii) absent when below 1050 AU/mL.
A rise in RBD-IgG concentration of 50% (1.5 fold) was considered as significant. Nucleoprotein-IgG was tested on specimens collected during the first sampling using the SARS-CoV-2 IgG assay (Abbott Diagnostics). Results were expressed as signal to cut-off ratio (S/CO). A nucleoprotein-IgG ratio of over 0.5 was retained as positive for a prior SARS-CoV-2 infection. Signed-ranked Wilcoxon test was used for comparison of IgG-RBD levels in the same participant. Spearman's non-parametric test was used to assess correlation between IgG-RBD levels after the first and second doses, and after SARS-CoV-2 infection and the first dose. The statistical significance threshold was set at 5%. Statistical analyses and graphs were performed using GraphPad Prism 9.1.1 (GraphPad Software, Inc., San Diego, CA).

Results
Demographic characteristics. The characteristics of the 396 residents included in the study are presented in Table 1 The RBD-IgG level after the first dose predicted the level after the second dose. In residents with prior SARS-CoV-2 infection, the first dose induced a high RBD-IgG level in most individuals (median; IQR: 24,617; 3909-43,692 AU/mL). RBD-IgG level was not significantly different after the second dose (23,341; 8112-50,341 AU/mL) (Fig. 1A). Twenty-eight residents (21.7%) had levels of below 1050 AU/mL after the first dose. Six participants (4.6%) failed to produce RBD-IgG levels of over 1050 AU/mL after the second dose, and two (1.5%) remained seronegative for RBD-IgG (< 50 AU/mL). A high RBD-IgG level after the first dose predicted the lack of RBD-IgG gain after the second dose (Fig. 1A).

RBD-IgG vaccine response impacted by both interval since infection and nucleoprotein-IgG status.
Residents infected during the first wave of the epidemic had a higher RBD-IgG response than those infected during the second wave (Fig. 3A,B). Conversely, the S/CO value of nucleoprotein-IgG was lower in residents infected during the first wave, compared to those infected during the second wave (Fig. 3C). Using the positive threshold retained for the study (S/CO: 0.5), detection of nucleoprotein-IgG was associated with a high RBD-IgG level after the first vaccine dose (PPV: 90.9%, NPV: 96.3%, sensitivity: 90.9%, specificity: 70.0%) (Fig. 3D). A lower sensitivity was observed when the threshold recommended by the manufacturer was used (S/ CO: 1.4), (PPV: 92.5%, NPV: 51.0%, sensitivity: 75.5%, specificity 80.6%) (Fig. 3D). Among residents with prior SARS-CoV-2 infection, the increase in RBD-IgG levels between the first and second doses was obvious in residents who tested negative for nucleoprotein-IgG, but not significant in those who tested positive (Supplemental Fig. 2). Thus, the first vaccine dose generated the best antibody response in residents who had been infected during the first wave and who continued to maintain nucleoprotein-IgG at the time of that vaccination (Fig. 3E). Multivariate logistic regression analyses were done to identify the independent determinants of RBD-IgG levels over 4160 AU/mL. We observed an independent association between first wave SARS-CoV-2 infection, the presence of N-IgG for infections occurring during the second wave, and a good response to the first and second vaccine doses (Table 2).   4A). A median decay of 4.5 fold was observed in IgG-RBD levels of NH residents tested at the two time points. Among NH residents for whom pre-vaccine antibody results were available, we found that the humoral vaccine response was associated with RBD-IgG level induced by natural immunization (Fig. 4B). A weak correlation was observed between RBD-IgG levels after natural immunization and the first vaccine dose (R 2 = 0.24, p = 0.03), (Supplemental Fig. 3A). A better correlation was observed between RBD-IgG levels 6-7 months after recovery and after the first vaccine dose (R 2 = 0.68, p = 0.002), (Supplemental Fig. 3B). The follow-up of RBD-IgG levels in 17 NH residents tested after natural immunization and vaccination is presented in Fig. 4C.

Discussion
Our study provides novel information on BNT162b2 vaccine response in older adults. Pre-vaccine SARS-CoV-2 infection, the interval passed since infection, and the persistence of a natural humoral immunity all influenced the antibody vaccine response. Assays that quantify anti-spike antibodies can be used on a large scale and involve a relatively low cost. Spike antibodies testing using automated assays constitutes a pragmatic approach to the assessment of vaccine response, and would constitute an advance towards a more tailored vaccine prophylaxis for NH residents. It has been previously reported that RBD-IgG levels after a single vaccine dose in persons under 65 with a documented history of SARS-CoV-2 infection were at least as good as those obtained after two doses in persons without prior infection [24][25][26] . Our study, conducted in NH residents in whom the humoral response was carefully assessed after one and two BNT162b2 vaccine doses, shows similar results overall. This suggests that the first vaccine dose elicits a sharp recall response, such as that in immunocompetent younger adults. In those NH residents, most often high responders after the first dose, the second dose did not significantly increase RBD-IgG, suggesting that older adults with prior COVID-19 infection may not systematically need a second vaccine dose. Delayed administration of the 2nd dose may be preferable for recently infected NH residents, as previously suggested 27 . In the absence of a recommendation to assess immunity following vaccination, the provision of at least two vaccine doses remains nonetheless justifiable, given that the second dose is safe and well tolerated.
Older frail individuals respond weakly to vaccination due to immunosenescence, affecting their capacity to produce antibodies after suffering influenza or receiving the first dose of SARS-CoV-2 vaccine 25,28 . The elderly show a clearly reduced response to the COVID-19 vaccine 29 . In residents who had not suffered SARS-CoV-2 infection, we previously observed a poor humoral response or a lack of RBD-IgG subsequent to the first BNT162b2 dose 11 . After two doses, the RBD-IgG response remained lower overall than in younger participants 23,25 . It has been reported that in health care workers and adults, two doses of the BNT162b2 vaccine induce a response comparable to that observed after one dose in persons with prior SARS-CoV-2 infection 9,30-32 . Furthermore, a third dose of BNT162b2 substantially reduced the rate of severe illness in people over 60 years who were exposed to the SARS-CoV-2 Delta variant 33 . No threshold is currently recognized for protection against Covid-19. However, several studies have reported data associating anti-Spike antibody levels with protection against infection. Based on data from phase 2/3 studies, a threshold of RBD-IgG at 1880 AU/mL (264 BAU/ mL) has been proposed for protection against symptomatic and asymptomatic SARS-CoV-2 Alpha variant infections 34 . This threshold was initially retained in France for persons identified as eligible for prophylaxis by monoclonal antibodies. Field data on health care workers 29 and NH residents 23 during the SARS-CoV-2 Alpha variant outbreak confirmed that such levels of RBD-IgG (1050 AU/mL and 993 AU/mL, respectively) correlate with COVID-19 protection. www.nature.com/scientificreports/ Vaccine response is influenced by various host factors, which also result in a heterogeneous vaccine response such as age, immunosuppression and prior infection 7,35 . However, prevention strategies could nonetheless be improved by the identification of poor vaccine responders among residents at risk of severe forms of illness. As for immunocompromised individuals, those residents would need additional vaccine doses. The risk of not producing a high vaccine response after a single dose was significantly higher in residents with prior SARS-CoV-2 infection who tested negative for nucleocapsid-IgG. Absent or low RBD-IgG responses are more frequent in asymptomatic and mild forms of SARS-CoV-2 infection 36 . In addition, the clearance of circulating antibodies against SARS-CoV-2 depends on the level reached after recovery 37 . We previously observed that 5% of confirmed COVID-19 cases tested negative for nucleoprotein-IgG 6-8 weeks after recovery 17,18 . In this study, almost a quarter of the NH residents tested negative for nucleoprotein-IgG 3-12 months after recovery. Testing nucleoprotein-IgG could be a useful means of predicting RBD-IgG response after the first vaccine dose, and for estimating the possible additional benefit of a second dose after natural immunization. Our results show that a modified threshold for nucleoprotein-IgG, of 0.5 S/CO instead of the 1.4 S/CO threshold proposed by the manufacturer, had a stronger predictive value for a high humoral response to the first dose. Our results also found that the second factor associated with a higher RBD-IgG response was a longer delay between infection and vaccination, confirming previous results 38,39 . Strong anamnestic responses require an interval of at least 4 months between primary and secondary immune responses 40 . Prime and boost vaccination generate different   www.nature.com/scientificreports/ plasma cells. Vaccine schedules aimed to induce long-lived plasma cells maintaining RBD-IgG levels at later time point. During the primary vaccination, short-lived plasma cells are generated with a rapid waning of antibodies, whereas the booster vaccination generates plasma cells providing long-lived immunity 41 . Spike antibodies are relatively easy to assess. The results of that assessment can be expressed in binding antibody unit/mL (BAU/mL), making comparisons of antibody levels easier. Despite such standardization, a definitive protective antibody level has yet to be determined. Hence, antibody testing is not currently recommended as a means of assessing immunity to SARS-CoV-2 following COVID-19 vaccination 42 . This study has several strengths. Our study is based on a large number of participants, probably representative of the population of NH residents in France. Nursing homes followed guidance published by our Health Agency (in line with EuGMS guidance 17,43 ), which explains that most NH residents with prior SARS-CoV-2 infection had PCR confirmation. Although NH residents have more comorbidities than most people of the same age, our results may also apply to frail older individuals living in the community. The main limitations of our study are the lack of a clinical outcome and the use of a unique mRNA vaccine (BNT162b2). Baseline antibodies prior to vaccination were only available for a small proportion of residents. Also, RBD-IgG levels were quantitated using an immunoassay, with concentration expressed in arbitrary units. We did not enumerate memory B cells and T cells that are critical for recall response against infection. In participants under 60 years of age who had recovered from SARS-CoV-2, the administration of a second vaccine dose close in time to the first showed no effect on the generation of memory B cells 44 . Neutralizing antibodies are considered a better marker of protection against severe form COVID-19, but such assays are not widely available in clinical practice. If neutralizing antibody levels appear to be a predictive marker of immune protection from severe COVID-19 37 , it remains to be demonstrated that RBD-IgG concentration is predictive of the level of protection against COVID-19.
Our study provides insight into the vaccination strategy involving the BNT162b2 vaccine. The need for a second dose 3 weeks after the first, in order to improve RBD-IgG response, is inconstant among NH residents with prior SARS-CoV-2 infection. NH residents who tested negative for RBD-IgG after the first vaccine dose are at high risk of producing a poor antibody response after two vaccine doses. For these participants, it remains to be determined whether a third dose may induce a significant RBD-IgG level. On the whole, these results suggest that consideration should be given to immunological monitoring in NH residents to optimize the vaccine response in this vulnerable population.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.